Earl W. Davie

Waterfall Sequence for Intrinsic Blood Clotting

A simple waterfall sequence is proposed to explain the function of the various protein clotting factors during the formation of the fibrin clot. When clotting is initiated, each cloting factor except fibrinogen is converted to a form that has enzymatic activity. This activation occurs in a sepwise sequence with each newly formed enzyme reacting with its specific substrate, converting it to an active enzyme.

Factor XI Deficiency in Ashkenazi Jews in Israel

BACKGROUND AND METHODS Severe factor XI deficiency, which is relatively common among Ashkenazi Jews, is associated with injury-related bleeding of considerable severity. Three point mutations--a splice-junction abnormality (Type I), Glu117----Stop (Type II), and Phe283----Leu (Type III)--have been described in six patients with factor XI deficiency. Clinical correlations with these mutations have not been carried out. We determined the relative frequency of the mutations and their association with plasma levels of factor XI clotting activity and bleeding, analyzing the mutations with the polymerase chain reaction and restriction-enzyme digestion. RESULTS The Type II and Type III mutations had similar frequencies among 43 Ashkenazi Jewish probands with severe factor XI deficiency; these two mutations accounted for 49 percent and 47 percent, respectively, of a total of 86 analyzed alleles. Among 40 of the probands and 12 of their relatives with severe factor XI deficiency, patients homozygous for Type III mutation had a significantly higher level of factor XI clotting activity (mean [+/- SD] percentage of normal values, 9.7 +/- 3.8 percent; n = 13) than those homozygous for Type II mutation (1.2 +/- 0.5 percent, n = 16) or compound heterozygotes with Type II/III mutation (3.3 +/- 1.6 percent, n = 23), as well as significantly fewer episodes of injury-related bleeding. Each of these three groups had a similarly increased proportion of episodes of bleeding complications after surgery at sites with enhanced local fibrinolysis, such as the urinary tract, or during tooth extraction. CONCLUSIONS Type II and Type III mutations are the predominant causes of factor XI deficiency among Ashkenazi Jews. Genotypic analysis, assay for factor XI, and consideration of the type and location of surgery can be helpful in planning operations in patients with this disorder.

Characterization of the Precursor of Prostate-specific Antigen ACTIVATION BY TRYPSIN AND BY HUMAN GLANDULAR KALLIKREIN

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-l-arginyl-l-prolyl-l-tyrosine-p-nitroaniline-HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by α1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.

Crystal structure of a 30 kDa C-terminal fragment from the γ chain of human fibrinogen

Abstract Background: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, ( α β γ ) 2 , held together by 29 disulfide bonds. The globular C terminus of the γ chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. Results: The X-ray crystallographic structure of the 30 kDa globular C terminus of the γ chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. Conclusions: The polymerization domain in the γ chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

A Brief Historical Review of the Waterfall/Cascade of Blood Coagulation

This article explores some of the events and people involved in unraveling the basic mechanisms leading to the clotting of blood. It also brings to focus the important role that my teachers and colleagues had on my career in research. When I entered college majoring in chemistry I had little idea that I would have an opportunity to become a professor of biochemistry at a major university. This was clearly the result of the excellent advice and encouragement that I received, particularly from my teachers early in my career. As a senior at the University of Washington, I took a course in biochemistry to complete my credit requirements for a B.S. in chemistry. The biochemistry course was taught by Donald Hanahan, an excellent lipid biochemist who understood the importance of a quantitative measurement. I was fascinated by the biochemistry class that Hanahan taught, as well as by a course dealing with the chemistry of natural products. When Hanahan invited me to work in his laboratory on a senior project, I got my first real experience of what it was like to do laboratory research and I enjoyed it. In the fall of 1950, I entered graduate school at the University of Washington and decided to do my thesis research with Hans Neurath to learn something about protein structure and function. Neurath was an excellent teacher, a distinguished protein chemist, and a leader in his field (Fig. 1). He also had high standards and was very demanding of his students. Neurath was originally trained as a colloid chemist at the University of Vienna. Following his postdoctoral training at the University of London in the chemistry department headed by Frederick Donnan, he immigrated to the United States in 1935, where his research interests shifted to protein structure. He then held positions at the University of Minnesota, Cornell University, and Duke University, and in 1950, he joined the University of Washington School of Medicine as chairman of a newly established Department of Biochemistry. His research interest then focused almost entirely on proteases, particularly pancreatic trypsin, chymotrypsin, and carboxypeptidase, because these proteins were available in sizable amounts and could be prepared in high purity. Much of his career then dealt with their structure and function, including their active sites, substrate specificity, their kinetics, amino acid sequence, interaction with inhibitors, and their mechanism of activation. Over the years, Neurath also became well known for his leadership in the publication of scientific literature, having founded and served as Editor-in-Chief of Biochemistry for 30 years. In 1990, he founded another new journal, Protein Science, as well as editing several excellent volumes, such as The Proteins initially with Kenneth Bailey and later with Robert Hill. For my Ph.D. thesis research, Neurath suggested that I should compare trypsinogen and trypsin to gain some insight as to the mechanism of zymogen activation. Little difference between the two proteins was anticipated because Cunningham and other postdoctoral fellows in Neurath’s laboratory had shown that trypsinogen and trypsin had essentially identical molecular weights of 23,800 as measured in the ultracentrifuge (1). In my initial studies, I was unable to detect any difference between the two proteins at their carboxyl-terminal end. This suggested that no peptides or amino acids were removed or peptide bonds cleaved at the THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 51, Issue of December 19, pp. 50819–50832, 2003

Amino acid sequence of human protein Z, A vitamin K-dependent plasma glycoprotein☆

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.

Nucleotide Sequences of the Three Genes Coding for Human Fibrinogen

Fibrinogen is synthesized in the liver by hepatic parenchymal cells and is secreted into the circulation (1). Hepatic synthesis of fibrinogen is constitutive but the rate can be modulated by a number of physiological and nonphysiological factors. The three chains of fibrinogen are encoded by distinct species of mRNA that are derived from the expression of three single copy genes (2, 3). Present evidence indicates that the three genes of human fibrinogen are linked and are located in a region that extends approximately 45 kb on chromosome 4q23-q32 (4). The genes are arranged in the order of γ-Aα-Bβ. The γ and Aα genes are transcribed in the same direction while the Bβ gene is transcribed in the opposite direction.

The Role of βγ and αγ Complexes in the Assembly of Human Fibrinogen

The role of αγ and βγ dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing β and γ chains and generating βγ complexes were fused with cells expressing only the α chain. Likewise, when cells co-expressing α and γ chains and generating αγ complexes were fused with cells expressing only the β chain, fibrinogen was also formed and secreted. The relative amounts of αγ or βγ intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the β chain was limiting, the αγ dimer was the predominant intermediate; likewise, when the α chain was limiting, the βγ complex was the predominant intermediate. The incorporation of preformed αγ and βγ complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of αγ and βγ complexes as functional intermediates in the assembly of fibrinogen.

[16] Human factor XII (hageman factor)

Publisher Summary In this chapter, a purification method of human factor XII is described that is reproducible and suitable for obtaining milligram amounts of protein. For purification frozen outdated human plasma is used, including following procedures: ammonium sulfate fractionation; first DEAE-Sephadex column chromatography; second DEAE-Sephadex column chromatography; QAE-Sephadex column chromatography; CM-celhdose column chromatography; and separation of Factor XII and Factor XII a by Benzamidine-Agarose column chromatography. The clotting activity of factor XII is determined by a kaolin partial thromboplastin time using human factor XII-deficient plasma. The clotting time is determined by continuous tilting of the tubes at 37°C. The units of factor XII activity are calculated from a standard calibration curve prepared by a serial dilution of pooled normal human plasma. Amidase activity of factor XII a is measured using the synthetic peptide substrate D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline.

ISOLATION, SUBUNIT STRUCTURE, AND PROTEOLYTIC MODIFICATION OF BOVINE FACTOR VIII*

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.